The vasculature of a normal adult is generally quiescent, with endothelial cells dividing approximately every 10 years; the formation of new blood vessels (angiogenesis) occurs only in a few physiological and pathological circumstances. In physiological circumstances, angiogenesis occurs during wound healing, organ regeneration, and in the female reproductive system during ovulation, menstruation, and the formation of the placenta. Angiogenesis occurs under pathological circumstances such as tumor, rheumatoid arthritis, diabetic retinopathy, psoriasis, and age-related macular degeneration (AMD). Recent evidence suggest that angiogenesis is the lifeline of solid tumor growth and metastasis. (Hanahan and Folkman, Cell 1996, 86:353-364.) Thus, anti-angiogenic therapy has become a hotly pursued field for the treatment of cancer and other disease related to angiogenesis.
Angiogenesis is subject to a complex control system consisting of multiple pro-angiogenic and anti-angiogenic factors. In adults, angiogenesis is tightly controlled by the balance between these factors. Vascular endothelial growth factor (VEGF) is an important pro-angiogenic factor, which regulates endothelial proliferation, permeability, and survival with high efficacy and specificity. (Folkman et al., Science 1987, 235:442; Giampietro et al., Cancer Metastasis Rev. 1994; Ferrara, Endocrine Rev. 2004, 25(4):581-611.) Thus VEGF and its signally pathway have become important targets for anti-angiogenic therapy in pathological conditions such as solid tumors, rheumatoid arthritis, etc.
There are three VEGF receptors, VEGFR-1 (fms-like tyrosine kinase, Ht-1), VEGFR-2 (fetal liver kinase 1-murine homologue/Kinase insert Domain containing Receptor-human homologue, KDR/Flk-1), and VEGFR-3 (Flt-4). VEGFR-1 and VEGFR-2 are expressed primarily on endothelial cells. VEGFR-3 is mainly expressed on lymphatic vessels and neuropilin, and is also expressed on neuronal cells. Each receptor has seven immunoglobulin-like domains in the extracellular domain, a single transmembrane region, and a consensus tyrosine kinase sequence. The receptor undergoes dimerization and ligand-dependent tyrosine phosphorylation in intact cells and results in a mitogenic, chemotactic and prosurvival signal. It has been demonstrated that VEGFR-2 is the major mediator of vascular endothelial cells (EC) mitogenesis and survival, as well as angiogenesis and microvascular permeability. (Ferrara N. et al., Nature Medicine 2003, 9(6):669-676.)
Blocking antibodies against VEGF or soluble VEGF receptor fragments can inhibit the binding of VEGF to VEGF receptors on vascular endothelial cells, thus block the VEGF-initiated signal transduction, and the pathological angiogenesis resulting from high VEGF expression. These angiogenesis inhibitors that have been developed as therapeutics include bevacizumab (Avastin), Lucentis, VEGF-Trap, etc. to treat angiogenesis-related diseases. Current anti-angiogenic drug Avastin of Genentech approved by FDA in 2004 is an example of specific anti-VEGF monoclonal antibody. (Ferrara et al., Nature Rev. Drug Disc. 2004, 3:391-400.) Its therapeutic mechanism is blocking VEGF-VEGF receptor interaction through binding to the VEGF molecule. However, it has two disadvantages: 1) relatively low binding affinity 2.3×10−9 leading to large doses; and 2) without inhibitory effect on placental growth factor (PIGF).
U.S. Pat. Nos. 6,100,071 and 5,952,199 describe VEGF binding fusion protein with Flt1 and KDR fragments, but these fusion proteins were not further developed because of low stability and serious side-effects. Although a fragment of Flt1 extracellular domain spanning the 2nd and 3rd immunoglobulin-like domains contains most of its binding activity to VEGF and PIGF, it has low effective activity in vivo because of its consecutive basic amino acids in the third immunoglobulin-like domain. Further, some therapeutics undergoing clinical trials, e.g., VEGF-Trap from Regeneron, have a stoichiometric ratio of 1:1 to VEGF molecules.
The present invention not only retains the binding activity of human Flt1 and KDR to VEGF and the binding activity to PIGF, but also provides enhanced bivalent interaction because of the increased distance between the two VEGF-binding immunoglobulin-like domains.